The present invention is directed to methods and devices for determining a spectroscopic characteristic of a sample utilizing laser induced fluorescence attenuation spectroscopy (“LIFAS”). More particularly, the invention is directed to methods and devices for measurement of the wavelength-dependent attenuation of the sample and subsequent restoration of the intrinsic laser induced fluorescence (“LIF”) for physiological monitoring, biological tissue characterization and biochemical analysis.
Conventionally, samples have been characterized by determining the attenuation and laser induced fluorescence (“LIF”). Once the attenuation and LIF of a sample have been determined, these spectroscopic properties can be utilized to determine a physical or physiological property of the sample. For example, the attenuation of a sample can be used to determine the concentrations of mixture components or turbidity of a fluid. Similarly, the LIF of a sample has been used in fields such as analytical chemistry, environmental monitoring, industrial inspection and medical diagnosis. In the medical field, for example, LIF spectroscopic techniques have been used for tissue characterization, malignant tumor identification, atherosclerotic plaque diagnosis, metabolism evaluation, and the like.
Conventionally, the attenuation or the absorption of a sample is determined by placing the sample between a light source and a detector and measuring any reduction in the intensity of the light as it passes through the sample. In order to obtain a measurement having an acceptable signal-to-noise ratio using these conventional techniques, it is important to transmit the incident light with sufficient intensity. Thus, the thickness of the sample and the wavelength of the incident light are important factors affecting the reliability of the resulting measurement. Moreover, because it is necessary to place the sample between the light source and the detector, it is difficult to perform attenuation measurements on certain types of samples, such as living tissue.
More recently, fiber optic techniques have been developed for the measurement of attenuation in which an optical fiber is used to guide the incident light to illuminate a sample inside a small chamber at the tip of a probe. A reflector is placed on the opposite side of the chamber to reflect the incident light into a second fiber which is associated with a detector. Unfortunately, these techniques find limited application with materials, such as fluids, that can readily pass into the chamber in the tip of the probe.
Conventionally, laser induced fluorescence (“LIFS”) spectroscopic techniques utilize various optical configurations in which a laser is directed at a sample using an optical fiber and the LIF from the sample is collected using a second optical fiber. Alternatively, the same fiber can be used for excitation of the sample and the collection of LIF. In either case, the LIF collected by the fiber is modulated by the sample, e.g. by the wavelength-dependent absorption and scattering of constituents of the sample. Therefore, existing LIFS methods are limited by the fact that the “intrinsic” or “true” fluorescence of the sample's fluorophores cannot be determined. Recent reports have suggested measuring the diffuse reflectance spectrum of the tissue as a means for correcting the intrinsic LIF using Monte-Carlo mathematical formulations. However, such correction methods are critically dependent on the backscattering characteristics of the tissue. Furthermore, backscattering does not account for the effects of absorption and scattering suffered by the intrinsic fluorescence prior to its measurement.
In biological LIF techniques, lasers are used to cause fluorophores in the sample to emit fluorescence. The main fluorophores in normal biological tissue are tryptophan, collagen and elastin. Other fluorophores, such as NAD (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), are also normally present, but at much lower concentrations. Under certain conditions, the contribution of the various fluorophores to the LIF can change. For example, during an ischemic or hypoxic event, the tissue is deprived of oxygen and anerobic respiration takes place. Consequently, the weak fluorophore NAD will be converted into the strong fluorophore reduced nicotinamide adenine dinucleotide (“NADH”). As a result, the LIF collected from the sample will reflect an increased contribution from NADH. This change is typically observed as a rise in the intensity of the LIF spectrum in the region of peak NADH emission, from about 470 to 490 nm. Thus, the metabolic state of the tissue can be determined by measuring the relative change in LIF intensity at the wavelength of peak NADH emission as compared to the relative change of the LIF intensity of fluorophores that are normally present in the tissue, such as elastin or collagen.
Unfortunately, the LIF of biological tissue is heavily modulated in the 390-450 nm range by the peak absorption of the main tissue chromophore, hemoglobin. Thus, although the intrinsic LIF spectra of normal tissue should approximately resemble that of the pure fluorophore components of the tissue, the measured LIF spectra has a valley in the 400-450 nm region that is associated with the hemoglobin absorption. As a result, the measured LIF spectrum tissue appears to have a double peak instead of the single peak spectrum associated with the pure fluorophores of the tissue. Hence, the LIF spectrum of normal tissue begins to resemble the spectrum associated with tissue suffering hypoxia or ischemia, which makes it more difficult to identify tissue abnormalities.
Furthermore, since the optical properties of biological tissue are influenced by its hemoglobin concentration, the measured LIF will vary with the level of blood perfusion throughout the cardiac cycle. The LIF of contractile tissue, such as the myocardium, is highly dependent on its state of contraction. Contraction increases the concentration of the fluorophore NADH and, hence, its contribution to tissue fluorescence. During contraction, however, blood is pumped out of the tissue, thereby reducing its hemoglobin concentration and, hence, light attenuation. Thus, a contracted myocardium retains less blood (i.e., a lower hemoglobin concentration) and, therefore, exhibits lower light absorption, than when relaxed.
Organs such as the brain, heart and kidney are the most sensitive to oxygen deficiency and can suffer permanent damage following an ischemic or hypoxic event. During open-heart surgery, for example, continuous monitoring of kidney perfusion, i.e., ischemia, is required. Similarly, since the success of a transplantation surgery is highly dependent on the level of organ perfusion at harvest and during preservation of the tissue, continuous monitoring of the organ is typically required.
Ischemia and hypoxia are both conditions that deprive tissue of oxygen, leading to anaerobic metabolism and the accumulation of the metabolic coenzyme NADH. The coenzyme NADH is a fluorescent molecule. Therefore, ischemia and hypoxia can be indirectly detected using LIF techniques by sensing increased concentrations of NADH and interpreting its elevation as a sign of oxygen deficiency. A common indicator of oxygen deficiency is the ratio between the LIF intensity at wavelengths associated with the peak fluorescence emission of NADH, collagen and elastin. However, such methods have not been practically applied for the detection of ischemia because of several complications. First, these methods cannot determine whether the elevated NADH concentration is caused by ischemia, hypoxia or hypermetabolism. Second, scarred or fibrosed tissue would be detected as normal because of their low NADH concentration. Finally, the indicator ratios are calculated by normalizing the intensity of NADH peak fluorescence by that of collagen or elastin. Although the fluoresence of the structural proteins elastin and collagen does not vary with tissue oxygenation, their fluorescence vary  varies with the site of measurement.
The intrinsic LIF of biological tissue has a single peak spectral profile resembling that of the pure structural proteins which are primarily elastin and collagen. However, this single peak profile is modulated by the attenuation of hemoglobin, especially about 410 nm, to yield a distorted, double-peak spectrum. The second spurious peak at about 470 nm results from the spectral valley created by heavy hemoglobin absorption at about 410 nm. Unfortunately, this second peak overlaps and obscures the peak fluorescence emission of NADH at about 470-490 nm, which may impede ischemia detection techniques that are based on NADH concentration.
The methods and devices of the present invention will enable measurement of several LIF parameters that directly relate to the state of tissue perfusion and/or hemoglobin oxygenation. This offers superior accuracy over the currently available LIF techniques which detect ischemia and hypoxia indirectly by sensing an upsurge in NADH fluorescence. In particular, the methods and devices of the present invention can directly indicate the presence and the level of hypoxia or ischemia. Furthermore, the methods of the present invention utilize novel criteria for discrimination between normal and ischemic or hypoxic tissue.
It is readily apparent that existing LIFS techniques in medicine and industry are affected by modulations of the intrinsic LIF. Thus, there is need for methods and devices for determining the intrinsic laser induced fluorescence while eliminating or minimizing modulating influences in order to provide more accurate and complete characterization of a sample. In particular, there is a need for method and devices for characterization of biological tissue. The present invention addresses these and other needs.